| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
release
Division of Pulmonary and Critical Care Medicine and Department of Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205-2196; and Department of Critical Care, Medical School of Athens University, Evangelismos Hospital, Athens, Greece GR106 76
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Kotanidou, Anastasia, Augustine M. K. Choi, Richard A. Winchurch, Leo Otterbein, and Henry E. Fessler. Urethan anesthesia protects rats against lethal endotoxemia and reduces TNF-
release. J. Appl. Physiol. 81(5):
2304-2311, 1996.
Urethan is a commonly used animal
anesthetic for nonrecovery laboratory surgery. However, urethan has
diverse biological effects that may complicate the interpretation of
experimental findings. This study examined the effect of urethan on the
response to an intravenous bolus of lipopolysaccharide (LPS; 30 mg/kg)
in rats. In instrumented rats, urethan (1.2 gm/kg ip) completely
prevented the fall in arterial pressure immediately after LPS
administration but did not prevent late cardiovascular collapse. In
uninstrumented rats, urethan also attenuated indexes of organ injury
measured 4 h after LPS administration, including mural bowel
hemorrhage, hemoconcentration, hypoglycemia, metabolic acidosis, and
lung myeloperoxidase activity, a measure of neutrophil sequestration.
The peak increase in tumor necrosis factor- (TNF-) 90 min after
LPS administration was reduced 88% by urethan (2,060 ± 316 vs.
16,934 ± 847 pg/ml; P < 0.001).
In uninstrumented animals, urethan at 1.2 gm/kg reduced the 90%
mortality rate of a lethal dose of LPS to 0-10% when
given up to 24 h before LPS administration but did not reduce mortality
when given 2 h after LPS. Urethan neither directly bound LPS by
Limulus assay nor inhibited
LPS-stimulated TNF- mRNA expression in cultured mouse peritoneal
macrophages, but TNF- mRNA expression was suppressed by serum from a
urethan-treated rat. Moreover, rauwolscine, which shares
2-adrenoceptor-blocking activity with urethan, also prevented death from a subsequent 90% lethal dose LPS bolus. We conclude that urethan or its metabolites protect against LPS, in part, by reducing TNF- release and
speculate that this may be mediated by
2-adrenoceptors. These actions
of urethan make it an undesirable anesthetic agent for in vivo studies of sepsis or LPS.
tumor necrosis factor- INTRODUCTION URETHAN (ethyl carbamate;
NH2COOC2H5)
is a trace component of alcoholic distilled spirits and is commonly
used as an animal anesthetic. A single intraperitoneal dose of
1-1.5 gm/kg produces hemodynamically stable anesthesia lasting up
to 12 h (6, 7). Urethan has a number of biological effects in addition
to anesthesia and analgesia. In rats, it causes leukopenia (16),
hypocalcemia (23), elevated corticosterone and epinephrine levels (21), and hyperglycemia (24). It also blocks
Despite these effects, urethan is frequently used for laboratory
anesthesia. We had been studying the effects of a bolus intravenous lipopolysaccharide (LPS) injection on hemodynamics in
barbiturate-anesthetized rats. When we changed to urethan anesthesia,
we serendipitously noticed a marked attenuation of the acute
hypotension after LPS administration. The present study was
undertaken to determine whether urethan modified the toxic effects of
LPS and to explore the mechanism of this pharmacological effect.
Because METHODS Animal Care
; lipopolysaccharide; septic shock; ethyl
carbamate; anesthetics; rauwolscine
2-adrenergic receptors (1).
Before its carcinogenicity was recognized (10), it was used briefly as
a hypnotic in humans and later as an anti-leukemic agent (17).
2-adrenoceptors on
macrophages can modulate tumor necrosis factor synthesis in vitro (25),
we speculated blockade of these receptors by urethan might modify the
response to LPS.
Acute In Vivo Experiments
Hemodynamic studies. The rats were anesthetized with intraperitoneal urethan (U; 1.2 gm/kg; n = 5) or thiobutabarbital (TBB; 80 mg/kg; n = 5), tracheotomized, and mechanically ventilated (model 683, Harvard Apparatus, South Natick, MA) with room air at a rate of 100 breaths/min and a tidal volume of 2 ml. PE-50 polyethylene catheters were placed in the internal jugular vein and common carotid artery. Arterial pressure (Pa) was recorded continuously with a physiological pressure transducer (Statham P23, Gould Electronics, Cleveland, OH) and a multichannel recorder (model 2800, Gould Electronics).After the surgery was completed, 30 min were allowed to ensure hemodynamic stabilization. The animals then received 30 mg/kg of LPS dissolved in 1 ml of saline via a tail vein over 1 min, and hemodynamic variables were monitored until death.
Biochemical and hematologic studies. A
separate set of 60 uninstrumented rats was studied. The rats received
1.2 gm/kg intraperitoneally of urethan dissolved in lactated Ringer
(RL) solution at 50 mg/ml (n = 30) or
an equivalent volume of Ringer solution alone
(n = 30). After 2 h, five rats from
each group were killed. Fifteen from each group of remaining rats then
received LPS intravenously (U+LPS and RL+LPS groups). Ninety minutes
after LPS administration, five rats from each of these two groups were
killed. All 40 remaining rats were killed 4 h after LPS administration
(U alone, U+LPS, RL alone, and RL+LPS groups;
n = 10 in each group). The rats were killed under halothane anesthesia by exsanguination from the abdominal aorta into a sterile heparinized syringe. Blood was immediately analyzed for glucose (Dextrostix reagent strips, Miles, Elkhart, IN),
arterial blood gases (ABL 30, Radiometer, Copenhagen, Denmark), and
spun hematocrit. Leukocyte and platelet counts were performed manually
with a hemocytometer. The remaining blood was centrifuged at 2,000 g, and the plasma was frozen at
70°C for subsequent measurement of tumor necrosis factor-
(TNF-) and interleukin-1. Blood from animals killed before and 90 min after LPS administration was only used for measurement of these
cytokines.
The abdomen was opened. Because urethan in higher concentrations has been reported to cause chemical peritonitis (30), peritoneal lavage was performed on animals not receiving LPS killed 2 and 6 h after Ringer solution or urethan administration. The bowels of all rats were examined for gross mural hemorrhage that was graded on a 0-4 ordinal scale as follows: 0, no visible hemorrhage; 1, <10% of bowel surface hemorrhagic; 2, 10 to <25% of bowel surface hemorrhagic; 3, 25 to <50% of bowel surface hemorrhagic; and 4, >50% of bowel surface hemorrhagic. The lungs were removed. The right lung was weighed wet, dried at 40°C to a constant weight, and reweighed to calculate the wet-to-dry weight ratio for use in the assay for myeloperoxidase (MPO). The left lung was frozen for later measurement of lung MPO activity, an index of polymorphonuclear leukocyte content (14).
MPO Assay
MPO was measured in lung tissue with a modification of the technique of Goldblum et al. (14). The lung was weighed and homogenized on ice with 5 ml of 50 mM phosphate buffer (pH 6). The homogenate was centrifuged at 4,000 g for 30 min, and the supernatant was discarded. The tissue pellet was resuspended in 5 ml of 0.5% hexadecyltrimethylammonium bromide and subjected to three cycles of freezing, thawing, and homogenation. The final homogenate was centrifuged at 4,000 g for 30 min. A 1.4-ml aliquot of assay buffer [0.1 ml of 0.3% H2O2, 17 mg o-dianisidine, and 50 mM phosphate buffer (pH 6) to make 100 ml] was added to 0.1 ml of supernatant, and the rate of change of absorbance at 460 nm was measured on a spectrophotometer (Beckman DU-64) with kinetics software. MPO activity is expressed as the change in absorbance units per minute per gram dry lung.TNF- Assay
was determined by an enzyme-linked immunosorbent assay
assay for mouse TNF- (Genzyme Diagnostics, Cambridge, MA), which
cross-reacts with rat TNF-. Fifty-microliter serum samples were
incubated in microwells coated with monoclonal anti-mouse TNF-.
After 2 h, the wells were decanted and washed, and 100 µl of
polyclonal anti-mouse TNF- conjugated to horseradish peroxidase were
added. After a 1-h incubation, the wells were again decanted and
washed. One hundred microliters of a peroxidase substrate solution
(tetramethylbenzidine) generating a colored product were added. After
10 min, the reaction was quenched by the addition of a stop solution,
and optical absorbance of each well at 450 nm was read in a
spectrophotometer (Beckman Instruments DU-64, Columbia, MD). Absorbance
was converted to TNF- concentration by comparison with a
simultaneously generated standard curve. The limits of detection of
this assay are 15-2,240 pg/ml, with inter- and intra-assay
variabilities of 7.1 and 3.4%, respectively (manufacturer's data).
Samples with a TNF- concentration exceeding the limits of the
standard curve were repeated after dilution.
Survival Studies
Rats weighing 200-250 g were anesthetized briefly with 5% halothane and injected intraperitoneally with varying doses of urethan. All doses were dissolved in lactated Ringer solution and diluted to a total volume of 6 ml/rat, yielding a maximal concentration of 50 mg/ml. This concentration has been previously shown not to induce peritonitis (30). Control animals received 6 ml of Ringer solution alone. The rats were reanesthetized with halothane to receive 30 mg/kg of LPS in 1 ml of Ringer solution via the dorsal penile or tail vein. The following groups were studied to examine the effects of urethan on survival: 1) 1.2 gm/kg of urethan followed after 2 h by LPS (n = 10); 2) 0.5 gm/kg of urethan followed after 2 h by LPS (n = 7); 3) 0.1 gm/kg of urethan followed after 2 h by LPS (n = 5); 4) lactated Ringer solution followed after 2 h by LPS (n = 10); 5) 1.2 gm/kg of urethan followed after 24 h by LPS (n = 5); and 6) LPS followed after 2 h by 1.2 gm/kg of urethan (n = 5).For comparison with another anesthetic, five animals were injected with
TBB (80 mg/kg ip), a long-acting barbiturate, followed after 2 h by
LPS. To examine the role of
2-adrenoceptor blockade, seven
rats were injected with 1 mg/kg of rauwolscine intravenously over 1 min, followed after 15 min by LPS. Finally, for comparison with a
structurally similar compound, rats were injected intraperitoneally with methyl carbamate (1 gm/kg, n = 6, or 2 gm/kg, n = 5), followed after 2 h
by 30 mg/kg of intravenous LPS. Methyl carbamate differs from ethyl
carbamate by a single methyl group but lacks anesthetic and
carcinogenic activities.
The animals were checked hourly for the first 8 h after LPS administration and then daily for a minimum of 7 days. The surviving animals were killed, and in those receiving urethan, the abdominal cavity was examined for adhesions or gross inflammation.
In Vitro Studies
Limulus lysate assay. The ability of urethan to directly bind and inactivate LPS was examined with the Limulus amoebocyte lysate assay. Briefly, 10
7 mg/ml of LPS
were incubated for 30 min with 2 mg/ml of urethan solution (2.25 × 102 M). One
hundred-microliter samples (n = 4) or
LPS standards were dispensed into pyrogen-free borosilicate tubes at
37°C. One hundred microliters of freshly reconstituted
Limulus lysate were added, and the
tubes were incubated for 10 min. Substrate (200 µl) was added, and
the incubation continued for an additional 3 min. The reaction was
stopped by the addition of 200 µl of 50% acetic acid, and the
absorbance was read at 405 nm. All samples and standards were run in
duplicate and averaged. Concentrations of the LPS standards ranging
from 108 to
107 mg/ml were plotted. The
concentrations of the samples incubated with urethan were calculated by
linear regression and expressed as percentages of the value of the
standard curve for 107
mg/ml of LPS.
TNF- mRNA Northern analysis. The
ability of urethan or serum from urethan-treated rats to directly
suppress LPS-induced TNF- mRNA expression was examined in cultured
peritoneal macrophages. RAW 264.7 murine macrophages (American Tissue
Culture Collection, Gaithersburg, MD) were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and 50 µg/ml of gentamycin. Cultures were
kept at 37°C in humidified 5%
CO2 in air. Subconfluent cultures
were treated for 30 min with either 2 mg/ml of urethan, its
diluent, 10 or 60% volumetric addition of serum from a rat treated 2 h
previously with 1.2 gm/kg of urethan intraperitoneally, or the same
volumes of serum from a control saline-treated rat. Cells were then
treated with 1 µg/ml of LPS (Escherichia
coli B8:127 or B5:05) for 3 h.
Total RNA was isolated by the STAT-60 RNAzol method with direct lysis
of cells in lysis buffer followed by chloroform extraction (Tel-Test B,
Friendswood, TX). Ten micrograms of total RNA were electrophoresed on a
1% agarose gel and transferred to a nylon membrane. The membranes were
prehybridized in buffer at 65°C for 2 h, followed by hybridization
in buffer containing 32P-labeled
murine TNF- for 24 h. Autoradiogram signals were quantified by
densimetric scanning (Molecular Dynamics, Sunnyvale, CA). Densimetric values for TNF- mRNA were normalized to simultaneously obtained values for 28S and 18S rRNA. This experiment was repeated in duplicate.
Reagents
Urethan, LPS from E. coli B8:127 and B5:055, hexadecyltrimethylammonium bromide, hydrogen peroxide, and o-dianisidine were obtained from Sigma Chemical, St. Louis, MO. Rauwolscine and TBB were obtained from Research Biochemicals International, Natick, MA. TNF- was measured
with the Factor-Test-X mouse TNF- enzyme-linked immunosorbent
assay assay kit from Genzyme Diagnostics, Cambridge, MA.
Statistical Analysis
The proportions of rats surviving 7 or more days in each group were compared by
2 analysis.
Dependent variables in the four groups of rats killed at 4 h were
compared by two-way analysis of variance, examining for effects of LPS,
urethan, and the interaction between LPS and urethan. Where significant
differences were detected, group means were compared by least
significant difference testing. The ordinally scaled bowel hemorrhage
in the four groups was compared by Mood median nonparametric testing.
Because the glucose reagent strips estimate glucose within one of seven
ranges, glucose levels were transformed to a 1-7 scale and then
compared by Mood median testing. Hemodynamic variables within each
group were compared against time by one-way analysis of variance for
repeated measures. Comparisons among groups were performed by two-way
analysis of variance, testing for effects of time and anesthetic. When
significant effects were found, individual time points with urethan and
TBB anesthesia were compared by least significant difference.
Statistical significance was assumed at = 0.05.
RESULTS
Hemodynamic Studies
All invasively monitored animals died within 4 h after LPS administration. Because of deaths occurring beyond 1 h, statistical analysis was limited to the first 60 min. The mean time of death was similar in both groups (U group, 102 ± 9.7 min; TBB group, 132 ± 30.6 min). However, the time course of the hemodynamic changes immediately after LPS administration was markedly different. The effects of LPS on mean Pa and heart rate (HR) for the groups of animals receiving urethan or TBB anesthesia are shown in Fig. 1. Baseline Pa and HR were not different between the two groups. After LPS administration, the TBB group became immediately hypotensive, followed by a partial recovery that was sustained until soon before death. HR was constant until soon before death. In contrast, the early hypotension after LPS administration did not occur in the U group. Pa remained relatively constant for 30 min and then declined gradually until death. HR increased after LPS administration and remained higher than in the TBB group until death.
P < 0.05. Significantly
different from corresponding time point under urethan anesthesia:
P < 0.001;
§ P < 0.05.
Biochemical and Hematologic Studies
Urethan attenuated LPS-induced changes in bloodborne elements, the gut, and the lung. Data are shown in Table 1. Four hours after LPS administration, hemoconcentration was present in both groups but was attenuated in the urethan-treated animals receiving LPS. As has been previously described (9, 16), both LPS and urethan caused leukopenia. However, the leukocyte count was not further reduced after LPS administration in the urethan-treated animals. Although the platelet count tended to be higher in the urethan-treated animals after LPS administration, urethan alone also caused a slight nonsignificant thrombocytosis. LPS caused a similar fall in the platelet count in both groups.
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
As has also been previously described, urethan alone elevated blood glucose (24) and LPS alone caused hypoglycemia (11). The blood glucose was reduced compared with urethan alone in the urethan-treated animals receiving LPS but remained higher than in the Ringer-treated animals after LPS administration. Calculated serum bicarbonate was reduced in the RL+LPS group and was normal in the other three groups.
In animals not receiving LPS, there was no difference in peritoneal leukocyte count 2 h after Ringer solution or urethan administration (RL group, 2,574 ± 1,086 cells/µl lavage; U group, 2,530 ± 935 cells/µl lavage). By 6 h, the peritoneal leukocyte count was lower in animals receiving urethan (3,034 ± 312 vs. 8,593 ± 1,530 cells/µl lavage; P < 0.05). There was no detectable bowel hemorrhage in the Ringer- or urethan-treated groups without LPS. Hemorrhage was extensive (mean value 2.9 on a 0-4 scale) after LPS administration in the Ringer-treated animals and tended to be less so (mean 1.7) in the U+LPS animals. In the lungs, neutrophil infiltration, as reflected by lung MPO activity, was not altered by urethan before LPS administration. MPO activity increased almost fourfold after LPS administration in the Ringer-treated animals. This increase was attenuated by pretreatment with urethan.
Serum TNF- levels were very low or undetectable before LPS
administration. The peak rise in TNF- 90 min after LPS
administration was attenuated eightfold by urethan pretreatment (Fig.
2). By 4 h after LPS administration,
TNF- levels were low in both groups, consistent with the known
kinetics of this cytokine (29, 32).
(TNF-) levels before
(time 0) and 90 and 240 min after
LPS administration in separate groups of animals pretreated with
urethan or diluent (Ringer solution). TNF- at 90 min is
eightfold lower in urethan-treated group
(P < 0.001). * Significantly
different from value at time 0 (P < 0.001).
Survival Studies (Fig. 3)
Nine of ten rats pretreated with Ringer solution alone died between 4 and 14 h after LPS administration, with a single rat surviving. In contrast, 9 of 10 rats receiving urethan at the 1.2 gm/kg dose survived, with the single death occurring on day 6 after LPS administration. All surviving animals were behaviorally normal within 7 days. This protective effect of urethan lasted at least 24 h because five of five animals injected with LPS 1 day after urethan (when they were lethargic but no longer unconscious) also survived. Urethan protection against LPS demonstrated a dose response because lower doses of urethan (which caused sedation but not surgical anesthesia) resulted in lower survival. Furthermore, the protective effect of urethan required that it precede the LPS. TBB, which produced >8 h of anesthesia, did not prolong survival after LPS administration, with five of five animals dying within 12 h. Five of six rats receiving 1 gm/kg of methyl carbamate and five of five rats receiving 2 gm/kg of methyl carbamate died within 48 h after LPS administration. Three additional rats treated with 2 gm/kg of methyl carbamate alone survived. All animals treated with the2-adrenoreceptor-antagonist
rauwolscine survived the lethal dose of LPS.
In Vitro Studies
To determine whether urethan bound LPS, LPS and urethan were coincubated for 30 min before assay for active LPS by Limulus lysate. There was no reduction in LPS activity by molar excess of urethan (LPS activity 102 ± 8% of control LPS activity; n = 4). To determine whether urethan directly suppressed TNF- expression by
macrophages, LPS-induced TNF- mRNA expression was measured by
Northern blot analysis in the presence and absence of urethan. Urethan,
in concentrations similar to those used in vivo, had no perceptible
effect on TNF- mRNA expression by mouse peritoneal macrophages
stimulated by LPS (Fig. 4). Likewise,
treatment of these macrophages with serum from a control rat had no
effect on the subsequent induction of TNF- mRNA. After incubation in a 60% volumetric dilution of control rat serum and LPS, induction of
TNF- mRNA was 98% of that in cells maintained in media and exposed
to LPS. However, incubation of macrophages with a 10 or 60% dilution
of serum from a urethan-treated rat attenuated LPS-stimulated TNF-
mRNA expression to 60 and 55%, respectively, of their levels in
media-treated LPS-exposed cells. Cells treated with B8:127 and B5:055
LPS responded quantitatively similarly.
mRNA in RAW 264.7 cells performed as
described in METHODS.
Lane 1, untreated cells;
lane 2, LPS-exposed cells;
lane 3, urethan-treated cells;
lane 4, urethan-treated LPS-exposed
cells. In contrast to its effects on serum TNF- in vivo, urethan
pretreatment did not inhibit TNF- mRNA induction by LPS.
DISCUSSION
Urethan is the ethyl ester of carbamate. Its hypnotic activity was recognized in the late 19th century, but it was little used for this purpose because its potency in humans was unpredictable. It was later recognized to be mutagenic and carcinogenic (13). Its current biological use is primarily as a long-acting anesthetic for animal surgery. Urethan produces eight or more hours of anesthesia after a single dose, during which time blood pressure and HR remain stable (7, 8). It is metabolized via the cytochrome P-450 system to carbon dioxide, ammonia, and water (13). It is rapidly cleared from the circulation, an intravenous dose being undetectable within 4 h.
Urethan has complex biological effects. It causes corticosterone
release in rats persisting for at least 24 h. This is believed to occur
via a central mechanism because it is suppressible by dexamethasone
(27). Urethan also causes epinephrine release, hyperglycemia,
lymphopenia, and neutrophilia (20, 32). Urethan blocks both central and
peripheral vascular
2-adrenoceptors (1) and
diminishes the pressor response to angiotensin (31) and the reflex tachycardia after hydralazine (22).
The present study of urethan demonstrates the following findings.
1) Urethan pretreatment prevents the
hypotension immediately after an intravenous LPS bolus.
2) Urethan attenuates LPS-mediated injury in the hematologic, gastrointestinal, and pulmonary systems. 3) Urethan reduces LPS-stimulated
TNF- release. 4) Urethan improves survival after a lethal LPS bolus.
5) Urethan neither directly binds
LPS nor blocks LPS-induced TNF- mRNA expression in vitro, but
TNF- mRNA expression is suppressed by serum from a urethan-treated rat. 6) Survival after LPS is also
improved by rauwolscine, which shares
2-adrenoceptor-blocking
activity with urethan. These findings lead us to speculate that the
2-adrenoceptor is coupled to
LPS-stimulated cytokine release and that urethan or its metabolites
improve survival and attenuate injury after LPS administration by
blocking this receptor.
TBB, a long-acting barbiturate, was used for comparison with urethan
because a single intraperitoneal dose provided anesthesia for the
duration of the hemodynamic studies. In TBB-anesthetized animals, we
found a time course of hemodynamic changes after LPS administration
similar to that which has been described previously in conscious
Sprague-Dawley rats (5). In addition, the hematologic and biochemical
changes after LPS administration in the conscious animals not receiving
urethan were fairly typical (9). The time course of TNF-
immunoreactivity was also similar to those found in previous studies
(9, 32).
Urethan treatment completely prevented the fall in Pa immediately after LPS administration. The cause of this early hypotension, which occurs before circulating inflammatory cytokines are detectable, is not known with certainty. It has been attributed to direct endothelial cell injury by LPS (20) and to nitric oxide (NO) (26). However, urethan does not appear to interfere with NO effects because the NO antagonist N-nitro-L-arginine causes equal increases in blood pressure in urethan-anesthetized and conscious rats (33).
It is unclear why there was no prolongation of survival in the surgically instrumented rats anesthetized with urethan because most uninstrumented rats treated with urethan survived the same dose of LPS. This may have been due to the additional physiological stress of surgery or of the volume-controlled mechanical ventilation.
Urethan attenuated several of the hematologic effects of LPS. In regard to platelet count, urethan alone tended to elevate the baseline value. LPS decreased platelets by an equal amount in urethan- and Ringer-treated animals. In contrast, urethan alone decreased the leukocyte count. This effect of urethan has been recognized for many years, but the mechanism is not known. Urethan completely prevented any further fall in leukocyte count after LPS administration. The peritoneal leukocyte count was unchanged by urethan at 2 h and was lower in urethan-treated animals after 6 h. This confirms that urethan in this dilute concentration did not cause the chemical peritonitis that has been described when it is used in higher concentrations (30). It also suggests that the attenuated response to LPS was not due to prior sequestration of leukocytes in the peritoneal cavity after urethan.
There was no difference in hematocrit in the urethan- and Ringer-treated animals not receiving LPS. This agrees with previous studies of urethan used in this concentration (30) and indicates similar initial plasma volumes in the two groups. LPS caused hemoconcentration in both Ringer- and urethan-treated animals but less so in the latter group. This suggests a reduction of systemic vascular leak with its resultant fluid extravasation.
Urethan pretreatment prevented death and reduced indexes of organ injury when administered 2 h before LPS. Lethality was reduced for as long as 24 h after urethan administration. Because urethan is rapidly cleared from blood, this suggests that the protective effect was due either to slow elimination of urethan from a tissue compartment or the activity of a metabolite.
Urethan also attenuated LPS-stimulated TNF- production. Although we
did not measure other inflammatory cytokines, we presume that the major
protective action of urethan was through its effects on TNF-.
TNF- injection reproduces many of the effects of LPS, including lung
injury, leukopenia, hypoglycemia, shock, acidosis, and death (18, 29).
Monoclonal antibodies against TNF- protect against LPS (3). The
finding that urethan fails to protect when injected 2 h after LPS
administration is also consistent with its major action being
suppression of TNF- because TNF- levels reach their maximum
earlier (9, 32). The inability of urethan to directly prevent TNF-
mRNA expression in vitro and the ability of serum from urethan-treated
animals to do so further suggest that it is a metabolite of urethan
rather than the parent compound that is producing this effect in vivo.
Alternatively, urethan may interfere with TNF- protein synthesis
posttranslationally or may induce synthesis of circulating
counterinflammatory cytokines such as interleukin-10.
Little previous work has studied how urethan alters the response to LPS. Consistent with the present study, Bibby and Grimble (4) found that urethan prevented the febrile response, the increase in hepatic zinc content, and the increase in blood urea nitrogen after low-dose (1.2 mg/kg) subcutaneous or intraperitoneal LPS. Foca et al. (12) administered very low dose LPS (0.64 mg/kg) intravenously to urethan-anesthetized rats. The dose of urethan was the minimum sufficient to alter Pa, and no comparison was made to conscious or otherwise anesthetized animals. This dose of urethan caused an immediate decrease in HR and a 20-30% fall in Pa, in contrast to the present study. However, after vagotomy and atropine, these urethan-anesthetized animals became hypertensive and tachycardiac when LPS was administered. This suggests the hypotensive/bradycardiac response to very-low-dose LPS was a vagally mediated reflex preserved under urethan anesthesia (12). This is not the mechanism of shock after lethal doses of LPS.
Protection against the lethality of LPS was also conferred by
pretreatment with rauwolscine, a highly specific
2-adrenoceptor blocker. This
suggests that at least some of urethan's effect resulted from blockade
of 2-receptors. Chlorpromazine,
which also has nonspecific -receptor-blocking activity, has also
been shown to reduce TNF- levels and prevent death after LPS
injection (13). Phenoxybenzamine, another nonspecific -adrenoceptor
blocker, was shown in early work to decrease LPS lethality in animals
(19). In more recent work, both phenoxybenzamine and idazoxan, a
blocker of 2- and imidazoline
receptors, decreased LPS-stimulated TNF- levels in mice (2).
2-Receptors have been
identified on murine cultured peritoneal macrophages. In that study,
norepinephrine or the specific
2-agonist UK-14304 augmented
LPS-induced TNF- production, and the augmentation was reversed by
yohimbine, another 2-receptor
blocker (25). In rabbit mesenteric arterial rings, in vivo LPS
injection inhibited contraction to electrical field stimulation. This
inhibition was reversed by yohimbine (28). Furthermore, because this
effect of in vivo LPS was reversed by in vitro yohimbine, it suggests
that 2-blockade may restore
vascular reactivity independent of the effects on TNF-. Yohimbine
also prevented the LPS-induced decrease in mesenteric blood flow and colonic motility in horses (8). Thus there are data from several models
indicating a pivotal role for the
2-receptor in the response to
LPS, mediating cytokine release and maintenance of vascular tone and
gut function.
The precise mechanism whereby
2-receptor blockade may reduce
the toxicity of LPS remains speculative and is probably multifactorial. Besides modulating TNF- production in macrophages,
2-receptors are present on a
wide variety of cells, including platelets, hepatocytes, pancreatic
islet cells, endothelial cells, vascular smooth muscle cells, and
central nervous system cells (27). In vascular smooth muscle, they are
present both as presynaptic negative-feedback autoreceptors that reduce
norepinephrine release and as postsynaptic or extrasynaptic receptors
on the myocytes that cause vasoconstriction in response to synaptic or
circulating catacholamines (27). Thus, in addition to suppressing
TNF-, there are numerous potential mechanisms whereby
2-adrenoceptor blockade could
ameliorate the effects of LPS on vascular tone, platelet aggregation,
and metabolism. Because we have not yet measured the effects of
2-adrenoceptor blockade on the
hemodynamic and cytokine responses to LPS, it remains uncertain whether
this fully explains the protective actions of urethan.
Several other actions of urethan may also contribute to survival after
LPS administration. Urethan increases circulating epinephrine levels
(21), which could preserve Pa. Urethan causes corticosteroid release.
Exogenous corticosteroids have been shown to reduce TNF- levels and
the lethality of LPS, and adrenalectomy markedly increases sensitivity
to LPS (15, 32). However, protection from LPS requires doses of
corticosteroids that greatly exceed endogenous levels during stress
or urethan anesthesia (15, 32). Thus it is unlikely that adrenocortical
hormone release explains the protection by urethan for LPS.
Given the complex and poorly understood activities of urethan, definitive explanation of its mechanism of action regarding LPS will require further study and will likely prove multifactorial. From a clinical perspective, it is unfortunate that the chemically similar but less toxic compound methyl carbamate was not protective. However, among the numerous related compounds, some may retain the protective actions of urethan without its carcinogenicity. These unique effects of urethan make it an inappropriate anesthetic agent for investigations of sepsis or other effects of LPS.
ACKNOWLEDGEMENTS
The authors are grateful for the secretarial assistance of Brenda Jordan, the suggestions and encouragement of Dr. Nicholas Flavahan, and the technical assistance of Camille Dickerson.
FOOTNOTES
Address for reprint requests: H. E. Fessler, Division of Pulmonary and Critical Care Medicine, Johns Hopkins Univ. School of Medicine, Ross Research Bldg., Suite 858, Baltimore, MD 21205-2196 (E-mail: hfessler{at}welchlink.welch.jhu.edu).
Received 21 December 1995; accepted in final form 17 June 1996.
REFERENCES
| 1. | Armstrong, J. M., F. Lefevre-Borg, B. Scatton, and I. Cavero. Urethane inhibits cardiovascular responses mediated by the stimulation of alpha-2 adrenoceptors in the rat. J. Pharmacol. Exp. Ther. 223: 524-535, 1982. |
| 2. | Bertini, R., S. Garattini, R. Delgado, and P. Ghezzi. Pharmacological activities of chlorpromazine involved in the inhibition of tumour necrosis factor production in vivo in mice. Immunology 79: 217-219, 1993. |
| 3. | Beutler, B., I. W. Milsark, and A. C. Cerami. Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science Wash. DC 229: 869-871, 1985. |
| 4. | Bibby, D. C., and R. F. Grimble. Effects of urethane, ambient temperature and injection route on rat body temperature and metabolism due to endotoxins. J. Physiol. Lond. 405: 547-562, 1988. |
| 5. | Brackett, D. J., C. F. Schaefer, P. Tompkins, L. Fagraeus, L. J. Peters, and M. F. Wilson. Evaluation of cardiac output, total peripheral vascular resistance, and plasma concentrations of vasopressin in the conscious, unrestrained rat during endotoxemia. Circ. Shock 17: 237-284, 1985. |
| 6. | Buelke-Sam, J., J. F. Holson, J. J. Bazare, and J. F. Young. Comparative stability of physiological parameters during sustained anesthesia in rats. Lab. Anim. Sci. 28: 157-162, 1978. |
| 7. | DeWildt, D. J., F. C. Hillen, A. G. Rauws, and B. Sangster. Etomidate-anaesthesia, with and without fentanyl, compared with urethane-anaesthesia in the rat. Br. J. Pharmacol. 79: 461-469, 1983. |
| 8. |
Eades, S. C.,
and
J. N. Moore.
Blockade of endotoxin-induced cecal hypoperfusion and ileus with an -2 antagonist in horses.
Am. J. Vet. Res.
54:
586-590,
1993.
|
| 9. |
Feuerstein, G.,
J. M. Hallenbeck,
B. Vanatta,
R. Rabinovici,
P. Y. Perera,
and
S. N. Vogel.
Effect of gram-negative endotoxin on levels of serum corticosterone, TNF, circulating blood cells, and the survival of rats.
Circ. Shock
30:
265-278,
1990.
|
| 10. | Field, K. J., and C. M. Lang. Hazards of urethane (ethyl carbamate): a review of the literature. Lab. Anim. 22: 255-262, 1988. |
| 11. | Filkins, J. D., and R. P. Cornell. Depression of hepatic gluconeogenesis and the hypoglycemia of endotoxin shock. Am. J. Physiol. 227: 778-781, 1974. |
| 12. | Foca, A., D. Altavilla, F. Mondello, D. Fumarola, and A. P. Caputi. Cardiovascular effects of Shigella sonnei endotoxin in the rat. Res. Commun. Chem. Pathol. Pharmacol. 38: 221-233, 1982. |
| 13. | Gadina, M., R. Bertini, M. Mengozzi, M. Zandalasini, A. Mantovani, and P. Ghezzi. Protective effect of chlorpromazine on endotoxin toxicity and TNF production in glucocorticoid-sensitive and glucocorticoid-resistant models of endotoxic shock. J. Exp. Med. 173: 1305-1310, 1991. |
| 14. | Goldblum, S. E., K. Wu, and M. Jay. Lung myeloperoxidase as a measure of pulmonary leukostasis in rabbits. J. Appl. Physiol. 59: 1978-1985, 1985. |
| 15. | Gonzalez, J. C., D. C. Johnson, D. C. Morrison, M. A. Freudenberg, C. Galanos, and R. Silverstein. Endogenous and exogenous glucocorticoids have different roles in modulating endotoxin lethality in D-galactosamine-sensitized mice. Infect. Immun. 61: 970-974, 1993. |
| 16. | Hawkins, J. A., and J. B. Murphy. The effect of ethyl urethan anesthesia on the acid-base equilibrium and cell contents of the blood. J. Exp. Med. 42: 609-618, 1925. |
| 17. | Hirschboeck, J. S., M. C. F. Lindert, J. Chase, and T. L. Calvy. Effects of urethane in the treatment of leukemia and metastatic malignant tumors. J. Am. Med. Assoc. 136: 90-95, 1948. |
| 18. | Kettelhut, I. C., W. Fiers, and A. L. Goldberg. The toxic effects of tumor necrosis factor in vivo and their prevention by cyclooxygenase inhibitors. Proc. Natl. Acad. Sci. USA 84: 4273-4277, 1987. |
| 19. | Lillehei, R. C., and L. D. MacLean. Physiological approach to successful treatment of endotoxin shock in the experimental animal. Arch. Surg. 78: 464-471, 1959. |
| 20. | Meyrick, B. O., U. S. Ryan, and K. L. Brigham. Direct effects of E. coli endotoxin on structure and permeability of pulmonary endothelial monolayers and the endothelial layer of intimal explants. Am. J. Pathol. 122: 140-151, 1986. |
| 21. | Ondo, J. G., and J. I. Kitay. Effects of urethan on pituitary-adrenal function in the rat. Proc. Soc. Exp. Biol. Med. 143: 894- 898, 1973. |
| 22. | Orallo, F., J. A. Fontenla, M. I. Loza, M. Campos, and J. M. Calleja. Effect of urethane on hydralazine-induced tachycardia in rats. Gen. Pharmacol. 23: 1143-1147, 1992. |
| 23. | Peng, T., C. W. Cooper, and P. L. Munson. The hypocalcemic effect of urethane in rats. J. Pharmacol. Exp. Ther. 182: 522-527, 1972. |
| 24. | Reinert, H. Urethane hyperglycaemia and hypothalamic activation. Nature Lond. 204: 889-891, 1964. |
| 25. |
Spengler, R. N.,
R. M. Allen,
D. G. Remick,
R. M. Strieter,
and
S. L. Kunkel.
Stimulation of -adrenergic receptor augments the production of macrophage-derived tumor necrosis factor.
J. Immunol.
145:
1430-1434,
1990.
|
| 26. | Thiemermann, C., and J. Vane. Inhibition of nitric oxide synthesis reduces the hypotension induced by bacterial lipopolysaccharides in the rat in vivo. Eur. J. Pharmacol. 182: 591-595, 1990. |
| 27. |
Timmermans, P. B. M. W. M.,
A. T. Chiu,
and
M. J. M. C. Thoolen.
-Adrenergic receptors.
In: Comprehensive Medicinal Chemistry. The Rational Design, Mechanistic Study & Therapeutic Application of Chemical Compounds, edited by P. G. Sammes,
and J. B. Taylor. Oxford, UK: Pergamon, 1919, p. 134-185.
|
| 28. | Tomikawa, S., and E. Okabe. Endotoxin impairs the response of rabbit mesenteric artery to electrical stimulation via a prejunctional mechanism. Br. J. Pharmacol. 107: 826-832, 1992. |
| 29. | Tracey, K. J., B. Beutler, S. F. Lowry, J. Merryweather, S. Wolpe, I. W. Milsark, R. J. Hariri, T. J. Fahey III, A. Zentella, J. D. Albert, G. T. Shires, and A. Cerami. Shock and tissue injury induced by recombinant human cachectin. Science Wash. DC 234: 470-474, 1986. |
| 30. | Van Der Meer, C., J. A. M. Versluys-Broers, H. A. R. E. Tuynman, and V. A. J. Buur. The effect of ethylurethane on hematocrit, blood pressure and plasma-glucose. Arch. Int. Pharmacodyn. Ther. 217: 257-275, 1975. |
| 31. | Volicer, L., and C. G. Loew. The effect of urethane anesthesia on the cardiovascular action of angiotensin II. Pharmacology 6: 193-201, 1971. |
| 32. | Waage, A. Production and clearance of tumor necrosis factor in rats exposed to endotoxin and dexamethasone. Clin. Immunol. Immunopathol. 45: 348-355, 1987. |
| 33. | Wang, Y., T. Zhou, T. C. Chua, and C. C. Y. Pang. Effects of inhalation and intravenous anaesthetic agents on pressor response to NG-nitro-L-arginine. Eur. J. Pharmacol. 198: 183-188, 1991. |
This article has been cited by other articles:
|
|
 |
|
  G. E. Hermann, C. A. Tovar, and R. C. Rogers LPS-induced suppression of gastric motility relieved by TNFR:Fc construct in dorsal vagal complex Am J Physiol Gastrointest Liver Physiol, September 1, 2002; 283(3): G634 - G639. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. D. Koblin Urethane: Help or Hindrance? Anesth. Analg., February 1, 2002; 94(2): 241 - 242. [Full Text] [PDF]
|
|
|
|
 |
|
 G. E. Hermann, G. S. Emch, C. A. Tovar, and R. C. Rogers c-Fos generation in the dorsal vagal complex after systemic endotoxin is not dependent on the vagus nerve Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2001; 280(1): R289 - R299. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. E. Hermann, C. A. Tovar, and R. C. Rogers Induction of endogenous tumor necrosis factor-alpha : suppression of centrally stimulated gastric motility Am J Physiol Regulatory Integrative Comp Physiol, January 1, 1999; 276(1): R59 - R68. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
Å · min
